Baculoviruses with enhanced virion production and a method for the production of baculoviruses

ABSTRACT

The present invention provides a method for restoring budding capability to GP64null baculoviruses including gp64null AcMNPV by expressing therein a portion of the VSV G protein gene or a truncated “stem” portion of the GP64 gene. Other embodiments provide methods to use portions of the G-stem or GP64 protein to target foreign proteins for display on virions.

REFERENCE TO RELATED APPLICATIONS

This application claims one or more inventions which were disclosed inProvisional Application No. 60/948,214, filed Jul. 6, 2007, entitled“BACULOVIRUSES WITH ENHANCED VIRION PRODUCTION AND A METHOD FOR THEPRODUCTION OF BACULOVIRUSES”. The benefit under 35 USC §119(e) of theUnited States provisional application is hereby claimed, and theaforementioned application is hereby incorporated herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.R01-A133657, awarded by the National Institutes of Health. The USGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to the field of virion production. Morespecifically, the invention pertains to methods for enhancing virionproduction in baculovirus strains, particularly GP64null baculoviruses,and a means to optionally express and display heterologous proteins onvirus particles or virions.

2. Description of Related Art

Baculoviruses are large double stranded DNA viruses that have beenstudied as agents for biological control of insect pests, as expressionvectors for high level production of heterologous proteins, and astransduction vectors and potential agents for human gene therapy. Thelatter applications derive from the observation that baculovirus virionscan efficiently enter a variety of human and other animal cell types anddeliver the baculovirus DNA genome to the nucleus of the cell. Viralentry is relatively efficient and promiscuous, permitting entry intomany different cell types that are not permissive for viral replication.Expression of foreign proteins in heterologous (non-permissive) cells isachieved by engineering the coding sequence of a foreign gene under apromoter that is active in the target cell type. For example, proteinexpression in human cells is achieved by placing the coding region ofthe foreign gene under the control of a promoter that is active in humancells, e.g. a human cytomegalovirus (HCMV) early promoter. Attempts toexpand the range of cells that are promiscuously entered by baculovirusAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV) virionsinclude studies in which the human Vesicular Stomatitis Virus envelopeglycoprotein known as G (VSV G) was expressed in addition to thebaculovirus envelope protein GP64. For many applications in gene therapyhowever, targeted entry of the baculovirus virion into specific celltypes would be highly desirable in order to either positively modify ornegatively affect the growth and/or survival of the target cell type.For example, cells infected with viruses such as HIV might be targetedfor destruction or death. Alternatively, genetic defects might becorrected by expression of a protein in specific cell types. Thus, theability to target baculovirus virion entry to specific cell types wouldbe of great value as a biotechnological tool in medicine or genetherapy. However, because of the promiscuity of AcMNPV entry intoheterologous cells, targeted entry is not currently possible with nativevirions.

AcMNPV is the baculovirus studied most extensively for gene therapyapplications. AcMNPV requires the major envelope glycoprotein known asGP64 for virion production and viral entry. In the absence of GP64,baculovirus virion production is severely reduced and the virions thatare produced are not infectious. Thus, GP64 is critical for theefficient production of virions and for the ability of those virions toenter host cells. Similarly, other baculoviruses such as LdMNPV, SeMNPV,or HaNPV require the so-called F envelope protein for entry. Studieshave shown that virions of the baculovirus AcMNPV can bepseudotyped—that is, the GP64 protein can be replaced with the envelopeprotein from another virus. AcMNPV viruses lacking GP64 (gp64null) thatexpress the human Vesicular Stomatitis Virus G (VSV G) protein are ableto produce infectious virions. However, like GP64, entry mediated by theVSV G protein is known to be highly promiscuous. Thus, VSV G does notprovide specificity in cell targeting. Studies pseudotyping AcMNPV withF envelope proteins from other baculoviruses showed that some but notall baculovirus F proteins could substitute for GP64. In addition,studies of baculovirus F proteins in pseudotyped retroviruses indicatethat they may not be useful in gene therapy applications as they did notmediate efficient entry into mammalian (mouse) cells.

The failure of gp64null baculoviruses to efficiently produce infectiousvirus particles is a critical and unresolved issue that must beaddressed in order to effectively use virions of baculoviruses likeAcMNPV in gene therapy applications where cell type targeting isnecessary or desirable. In addition to gene therapy applications, it maybe desirable to display foreign proteins on the surface of a gp64nullvirion for other purposes, such as production of a therapeutic or avaccine. With existing methodology, it is difficult to produce usablequantities of gp64null baculovirus particles containing heterologousenvelope proteins. This problem presents a substantial constraint on thepotential use of pseudotyped gp64null viruses. The present invention isdirected to solving this problem and other problems that are associatedwith the production of virions or pseudotyped virions.

SUMMARY OF THE INVENTION

The present invention addresses the issue of inefficient budding fromgp64null viruses. The following strategy was used: A membrane proximalfragment of the VSV G protein (hereinafter referred to as a G-stem) wascloned into the genome of a gp64null baculovirus. In a preferredembodiment of the invention, the G-stem polypeptide includes amino acidsequences from each of: the C-terminal portion of the ectodomain; thepredicted transmembrane domain; and the predicted cytoplasmic domain. Ina more preferred embodiment of the invention, the G-stem polypeptideincludes a sequence of 20-130 amino acids from the C-terminal portion ofthe ectodomain; substantially all of the predicted transmembrane domain;and a sequence of from about 10 to substantially all of the amino acidsthat make up the predicted cytoplasmic tail domain. In another preferredembodiment of the invention, the VSV G stem polypeptide includes: about42 amino acids from the C-terminal portion of the ectodomain; about 28amino acids from the predicted transmembrane domain; and about 21 aminoacids from the predicted cytoplasmic tail domain.

In addition to overcoming the budding defects, the methods of thepresent invention provide a means for displaying foreign proteins on thesurface of gp64null baculovirus virions. Chimeric fusion proteinscontaining a G-stem polypeptide or a portion of GP64 can be targeted togp64null virions produced by G-stem stimulated budding. In a preferredembodiment of the invention, these chimeric proteins contain aheterologous protein or protein fragment that confers cell-specifictargeting and entry.

Virions which express heterologous proteins as produced by the methodsof this invention can be used for a variety of purposes including:

1. Gene therapy targeted to one or many cell types.

2. Vaccine production where the display of a heterologous protein on thesurface of a baculovirus particle elicits a more robust immune response.

3. Other applications involving display of a membrane or other proteinon virus particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a strategy for generation of a gp64-null AcMNPV bacmid byhomologous recombination in E. coli.

FIG. 1B shows a strategy for insertion of a VSV G-stem construct intothe polyhedrin locus of the gp64null AcMNPV bacmid.

FIG. 2 shows a strategy for rescuing virion production by gp64nullbaculovirus infected cells under several conditions.

FIG. 3A shows a Western blot of purified virions challenged with ananti-cMyc antibody, and demonstrating rescue of virion production byG-stem expression.

FIG. 3B shows a Western blot of purified virions as in FIG. 3Achallenged with an anti-cMyc antibody and an anti-VP39 antibody.

FIG. 3C shows the blot from FIGS. 3A and 3B (which contains ³⁵S-metlabeled purified virions) imaged on a PhosphorImager™ screen.

FIG. 3D shows a Western blot of purified virions challenged with ananti-VSV-G antibody.

FIG. 3E shows a Western blot of purified virions challenged with ananti-VSV-G antibody and an anti-VP39 antibody.

FIG. 4A shows a quantitative comparison of progeny budded virion (BV)production from Sf9 cells infected with several baculovirus constructs(vAc^(gp64−/Acgp64), vAc^(gp64−) and vAc/G-Stem).

FIG. 4B shows quantitative comparisons of the VP39 protein band as anindicator of budded virus production from various preparations as shownin FIG. 4A.

FIG. 5A shows a strategy for insertion of VSV G-Stem fusion protein geneconstructs into the polyhedrin locus of a gp64null AcMNPV bacmid.

FIG. 5B shows representative G-stem fusion constructs and Western blotanalyses of products from gp64null baculoviruses expressing each.

FIG. 5C shows fluorescence micrographs of cells infected with anEGFP-G-stem construct.

FIG. 6A shows a strategy for constructing a series of cMyc tagged GP64constructs that are truncated at the N-terminus of the GP64 ectodomain.

FIG. 6B shows Western blot analysis of cell extracts from cells infectedwith gp64null viruses expressing the GP64 constructs shown in FIG. 6A(left panel) or co-infected with the viruses expressing the constructsfrom FIG. 6A and a virus expressing the VSV G-stem construct (rightpanel).

FIG. 6C shows Western blot analysis of purified budded virions fromcells infected with viruses expressing the GP64 constructs shown in FIG.6A (left panel) or co-infected with the viruses expressing theconstructs from FIG. 6A and a virus expressing the VSV G-stem construct(right panel).

FIG. 6D shows the strategy for constructing a series of cMyc taggedtruncated GP64 constructs. A series of constructs containing the GP64“stem” domain and various portions of the GP64 ectodomain weregenerated.

FIG. 6E shows Western blot analysis of cell extracts from cells infectedwith viruses expressing the G-stem construct or the GP64-stem fusionsalone (left panel) or co-infections of viruses expressing the GP64-stemfusions and a virus expressing the VSV G-stem construct (right panel).

FIG. 6F shows Western blot analysis of purified budded virions (BV) fromcells infected with viruses expressing GP64-stem fusions (left panel) orco-infected with viruses expressing the GP64-stem fusions and a virusexpressing the VSV G-stem construct (right panel).

FIG. 6G shows a diagrammatic representation of strategies used forinfection or co-infection of Sf9 cells with viruses expressing fusionproteins containing the GP64-stem and display of the GP64-stem fusionson BV.

FIG. 7 shows strategies for display of heterologous proteins on thesurface of gp64null AcMNPV budded virions.

FIG. 8A shows a strategy for generating HA-GP64 fusions and insertion ofchimeric HA constructs into the polyhedrin locus of a gp64null AcMNPVbacmid.

FIG. 8B shows Western blot analysis of BV preparations from virusesexpressing HA-GP64 fusions and co-infection with a G-stem expressingvirus.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, baculoviruses such as AcMNPV containing a knockout inthe gp64 gene are unable to produce virions efficiently in the absenceof the GP64 protein and this represents a major obstacle to the use ofgp64null viruses in research and biotechnology. The present inventionprovides a method to solve this and related problems.

The present invention provides methods for restoring efficient buddingcapability to GP64null baculoviruses including gp64null AcMNPV byexpressing therein or otherwise providing a portion of the VSV G proteinor a portion of GP64. Other embodiments provide methods to expressforeign proteins on virions. By choosing stem constructs of theinvention and including appropriate heterologous proteins that confercell-specific targeting and entry, the invention provides a means forproduction of gp64null baculovirus virions capable of cell-specificentry. The use of G-stem or GP64-stem constructs as identified here, aschimeric fusions with heterologous proteins also provides a means oftargeting proteins to the virion membrane or envelope. In addition, themethods of the present invention provide a means for vaccine productionwherein the expression of a heterologous protein on the surface of abaculovirus particle elicits a more robust immune response. Finally, theinvention enables other diverse applications as are well known to thoseskilled in the art where expression of a membrane or other protein onviral particles is desirable.

Although the VSV G protein and the GP64 proteins are discussed herein,stem constructs derived from envelope glycoproteins of otherbaculoviruses or other members of the Rhabdovirus family ofnegative-sense RNA viruses are also within the spirit of the presentinvention.

It was not obvious that VSV G-stem polypeptides would provide a solutionto problems encountered with gp64null baculovirus budding. Indeed,several factors suggested that a VSV G-stem would not be compatible withbaculoviruses. These factors include: baculoviruses (large DNA viruseswith genomes of 100+ genes that replicate in the nucleus) are completelyunrelated to rhabdoviruses like VSV (negative stranded RNA viruses withsmall genomes of only 5 open reading frames (ORFs) that replicate in thecytoplasm). In addition, VSV proteins bear no obvious amino acidsequence similarity to those of Baculoviruses and the virion structureof the two viruses appears to have little in common beyond superficialfeatures of enveloped viruses. Despite these considerations, expressionof G-stem constructs during infection by a gp64null baculovirusaccording to the methods of the present invention resulted in theefficient production of virions and thus rescued the severe buddingdefect observed in the gp64null virus.

In one embodiment of the invention, a truncated VSV G gene construct(referred to generically herein as G-stem constructs) was created andthe gene was inserted into the AcMNPV baculovirus genome under thecontrol of the baculovirus AcMNPV gp64 promoter. In a preferredembodiment of the invention, such VSV G gene constructs included anAcMNPV gene promoter and signal peptide, an epitope tag, and anN-terminally truncated VSV G gene fragment. An exemplary VSV G geneconstruct of the invention includes the AcMNPV gp64 promoter and signalpeptide, a cMyc epitope tag (at the N-terminus of the mature protein),42 amino acids from the VSV G ectodomain (positions 421 to 463), plusthe predicted transmembrane (TM) and the cytoplasmic tail (CTD) domainsof VSV G.

This G-stem construct was inserted into an AcMNPV bacmid containing agp64 deletion, using a method described in Lung et al., 2002, J. Virol.76, pages 5729-5736, herein incorporated by reference. Construction ofthe baculovirus bacmid is summarized in FIGS. 1A and 1B. FIG. 1A shows astrategy for generation of a gp64-null AcMNPV bacmid by homologousrecombination in E. coli. The gp64 locus of the AcMNPV bacmid(bMON14272) is shown above a fragment that was used as a transfer vectorto replace the gp64 locus with a chloramphenicol resistance gene (cat).Sequences included for homologous recombination (107325 to 108039 and109761 to 112049) are indicated.

FIG. 1B shows a strategy for insertion of a VSV G-stem construct intothe polyhedrin locus of the gp64null AcMNPV bacmid. The cassetteinserted into the gp64null bacmid includes a p6.9 promoter-GUS reporterplus sequences encoding a truncated VSV G protein (VSV G-stem) under thecontrol of a gp64 promoter (64pro). The VSV G-Stem construct encodes thegp64 signal peptide, followed by a cMyc tag and a truncated version ofthe VSV G protein that encodes 42 amino acids of the C-terminal portionof the VSV G ectodomain, plus the transmembrane (TM) and cytoplasmictail domains (CTD) of VSV G. Numbers above the cMyc-G-Stem cassette(421-511) indicate amino acid sequence numbers from the VSV G protein.G-Stem fusion protein gene cassettes were inserted into the polyhedrinlocus of a gp64null AcMNPV bacmid by Tn7-based transposition.

The resulting bacmid was used to generate a virus, designatedvAc/G-Stem, by transfecting the bacmid into a stable cell line thatconstitutively expresses a wild type GP64 protein. One such cell linesuitable for the invention is known as Sf9^(Op1D). The strategy forpropagation of the virus is summarized in FIG. 2.

FIG. 2 shows a strategy for rescuing virion production by gp64nullbaculovirus infected cells under several conditions. The diagram showsstrategies for producing budded virions from gp64null viruses, usingeither a cell line (Sf9^(Op1D)) or a VSV G-stem construct. Morespecifically, a baculovirus genome contains a deleted gp64 gene (gp64knockout, vAc⁶⁴⁻), and that baculovirus can be propagated in a stablecell line expressing OpMNPV GP64. When the GP64 null virus infectsSf9-Op1D, the cell line expressing OpMNPV GP64, the virus without thegp64 gene can bud and propagate, thus resulting in budding of a GP64nullvirus with OpMNPV GP64. When the GP64 null virus infects SF9 cells, thevirus can enter the cells and partially replicate, but virion budding isdefective, and results in only about 2-5% budded virions lacking GP64.

Alternatively, a gene encoding the “stem” region of the VSV G protein isinserted into the viral genome containing the gp64 knockout(vAc/G-Stem), and then propagated in the cell line expressing OpMNPVGP64. When the GP64null/G-stem+ virus is then used to infect Sf9 cells,virion budding is restored, resulting in budded virions displaying theVSV G stem.

To determine if a G-Stem had an effect on virion budding, an AcMNPVbudding assay was performed. Sf9 cells were infected with control orG-Stem expressing gp64null viruses (previously generated and titred inSf9^(Op1D) cells). Next, progeny virions were metabolically labeled with³⁵S-methionine. Supernatants containing labeled progeny virions werecollected and virions were purified by pelleting through 25% sucrose. Inthis budding assay, budded virions are isolated from the supernatant andonly progeny virions are labeled. Progeny virions were isolated in thismanner from Sf9 cells infected with viruses vAc^(gp64−) (a gp64nullvirus), vAc^(gp64−/Acgp64) (a gp64null virus that was repaired byreinserting a gp64 gene), and vAc/G-Stem (a gp64null virus thatexpressed a G-Stem construct).

FIGS. 3A-3E shows Western blot analyses of purified virions produced bygp64null baculovirus under several conditions. The Western blot analysesusing both anti-cMyc and anti-VSV G antibodies show G-Stem protein inpurified budded virions from the cells infected with the vAc/G-stemvirus.

The Western blot was challenged with an anti-cMyc antibody and analyzed(FIG. 3A), then the same blot was challenged with an anti-VP39 antibodyand analyzed again (FIG. 3B). In addition, the same blot was imaged on aPhosphorImager™ screen (FIG. 3C) to detect labeled virion proteins.Because VP39 (the major capsid protein) is highly abundant in virionsand the quantity of VP39 per virion appears to be constant, the VP39protein in virion preparations is typically used as an indicator ofrelative virion quantity. FIG. 3D shows a Western blot challenged withanti-VSV G and FIG. 3E shows a Western blot challenged with anti-VSV Gand anti-VP39. Lane 1 of FIGS. 3A-3E and FIGS. 4A-4B shows buddedvirions purified from Sf9 cells infected with vAc^(gp64−/Acgp64). Lane 2shows budded virions purified from Sf9 cells infected with vAc^(gp64−)and lane 3 shows budded virions purified from Sf9 cells infected withvAc/G-Stem. The open circles in these figures indicate the position ofthe major capsid protein, VP39, of AcMNPV and arrowheads indicate thepositions of the cMyc-tagged G-stem protein, as detected by therespective antibodies. Purified virions were examined by SDS-PAGE andWestern blotting.

FIG. 4A shows a quantitative comparison of budded virion (BV) productionfrom Sf9 cells infected with several baculoviruses: vAc^(gp64−/Acgp64),vAc^(gp64−) and vAc/G-Stem. More specifically, FIG. 4A shows aPhosphorImager™ analysis of purified labeled virions. Sf9 cells wereinfected with each virus, labeled with ³⁵S-Methionine, and progenyvirions were isolated from infected cell supernatants by centrifugationof the supernatant through a sucrose cushion, followed by separation ofvirions on equilibrium sucrose density gradients. Virions derived fromequivalent amounts of infected cell culture supernatants were loadedonto SDS-PAGE gels and examined by phosphorImager™ analysis of labeledvirion proteins.

Quantitative comparisons of ³⁵S-Methionine labeled VP39 bands frombudded virion preparations derived from equivalent quantities of cellsupernatants indicated that virion production in the gp64null virusexpressing a G-stem construct (virus vAc/G-Stem) was approximately 2.27times higher than that of the virus expressing wild type GP64, and about11 times higher than that detected from the gp64null virus (FIGS.4A-4B). FIG. 4A is one representative example from among 3 sets ofindependently labeled budded virion profiles and the graph in FIG. 4Bwas generated with data from three independently labeled viral profiles.FIG. 4B shows quantitative comparisons of the VP39 protein band fromvarious preparations as shown in FIG. 4A. Quantification data representaverages and standard deviations derived from three independentlylabeled BV preparations.

These data show that expression of a VSV G-Stem construct in the contextof a gp64null baculovirus resulted in the rescue of the severe buddingdefect caused by the absence of the GP64 protein. Indeed, preliminarymeasurements suggest that budding stimulated by a G-stem construct mayeven exceed that from a virus expressing the wild-type GP64 protein.Thus, using the methods of the present invention, budded virions thatcontain no native GP64 protein can be efficiently generated. It isfurther possible to produce virions that express foreign proteins in theabsence of a native GP64 protein using these methods. Such virions haveimportant applications in biotechnology, including applications invaccine development and gene therapy.

In another embodiment, expression of the G-stem construct under astronger promoter such as the AcMNPV polyhedrin or p10 promoter mayresult in even higher levels of GP64null virion production.

A similar system is applicable to related baculoviruses that carry an Fprotein and no GP64 protein, in the following manner. The F gene isdeleted from the genome of a virus such as a group II NPV or GV, bymethods similar to those used for deleting the gp64 gene from AcMNPV,using a cell line engineered to express the F protein or a suitablehomolog. Budding by the resulting F-null virus is then rescued byproviding either a G-stem construct, a GP64-stem construct, or a similarportion of the F protein. Additionally, heterologous peptides orproteins may be displayed by generating fusions with the stem region ofthe homologous F protein. Alternatively, heterologous proteins (GP64,VSV G, etc.) could be used as a source for the stem regions that areused for generating fusions and targeting the proteins to the virion.

Several embodiments using methods of the present invention to expressforeign proteins on the surface of gp64null AcMNPV virions are disclosedherein. A first embodiment includes expression of one or more nativemembrane or envelope proteins in combination with a G-stem construct ingp64null virus infected cells. A second embodiment of the inventionincludes expression of protein fusions containing all or a portion ofthe ectodomain of a foreign protein fused to a G-stem construct. A thirdembodiment of the invention includes the expression of protein fusionscontaining all or a portion of the ectodomain of a foreign protein fusedto a portion of the GP64 protein including the transmembrane andcytoplasmic domains of GP64. A fourth embodiment uses one or moreportions of other baculovirus virion membrane proteins (for instanceAc23 or F proteins from other baculoviruses) as the “stem” region forfusions with one or more foreign proteins. In the second and thirdembodiments above, a G-stem construct alone could be expressedseparately to provide the budding function. Alternatively in otherpreferred embodiments, foreign proteins fused to a G-stem providesufficient rescue of budding in the absence of a separately expressedG-stem construct.

As examples of the embodiments described above, a series of G-stemfusion constructs was generated. A G-stem as described previously wasfused to the C-terminus of either the enhanced green fluorescent protein(EGFP) or various portions of the GP64 ectodomain. These G-stem fusionconstructs were then inserted into a gp64null baculovirus. The strategyfor generating these constructs is outlined in FIG. 5A. FIG. 5A shows astrategy for insertion of VSV G-Stem fusion protein gene constructs intothe polyhedrin locus of a gp64null AcMNPV bacmid. Constructs weregenerated to express peptides or proteins fused with a VSV G-Stem andunder the control of the gp64 promoter (64pro). Peptides fused to theG-stem included the EGFP protein as well as portions of the AcMNPV GP64protein. Peptides derived from the GP64 protein (designated 130, 152,160, 166, and 175) were fused to the G-Stem fusion protein. The cassetteinserted into the bacmid also included a p6.9 promoter-GUS reporterconstruct.

Expression and analysis of G-Stem fusion proteins by Western blotanalysis and immunofluorescence microscopy are illustrated in FIGS.5B-5C. FIG. 5B shows representative G-stem fusion constructs and Westernblot analyses of products from gp64null baculoviruses expressing each.Cell extracts or purified BV preparations derived from Sf9 cellsinfected with the gp64null viruses expressing G-stem fusion constructswere examined for protein expression using Western blot analysis with ananti-cMyc antibody. All G-stem constructs contained a cMyc epitope tag.Analysis of cell extracts shows that each G-stem construct was stablyexpressed in the infected cells, and analysis of purified BV shows thatvarious G-stem constructs were targeted to and assembled into the BV.FIG. 5C shows fluorescence micrographs of cells infected with anEGFP-G-stem construct. Micrographs show fluorescence from infected cellsat 24 and 48 hours post infection.

In a preferred embodiment of the invention, G-stem polypeptides werefused to peptides of about 130 to 175 amino acids in length (derivedfrom the GP64 ectodomain), or fused to EGFP, then cloned under the GP64promoter and inserted into a gp64null AcMNPV virus genome. The resultingviruses were propagated in appropriate host cells such as Sf9^(Op1D)cells then used to infect Sf9 cells. Expression of the fusion proteinswas detected by Western blot analysis of cell extracts or purified BVusing an anti-cMyc antibody. All fusion constructs were expressed wellin infected Sf9 cells (FIG. 5B, upper panel, lanes 2-7). Examination ofcells infected with the virus containing the EGFP-G-stem fusion byimmunofluorescence microscopy (FIG. 5C) further demonstrated expressionof that construct in the infected cells. All G-stem fusions tested,except a fusion containing a 175 amino acid region from GP64, weredetected in purified preparations of BV (FIG. 5B, lower panel). TheG-stem construct alone (unfused) mediated robust budding (FIGS. 3A-3Eand 4A-4B) and was found in abundance in the BV and at higher levelsthan that observed with the fusion constructs (FIG. 5B, lower panel;compare lane 1 to 2-7, arrow). Using the single cMyc epitope present oneach construct, these data show clear relative differences in detectionof the different G-stem fusion constructs and confirm that most (all butone) of these fusion constructs were a) capable of mediating virionbudding, and b) targeted to, and displayed on gp64null virions. Thus,because most of the G-stem fusion protein constructs evaluated werefound in the purified virion preparations, these data show that this isa viable strategy for generating BV and displaying foreign proteins onAcMNPV gp64null virions.

In an example of those embodiments of the invention that include theexpression of protein fusions containing all or a portion of thetransmembrane and cytoplasmic domains of the GP64 protein, a series ofbaculoviruses that expressed proteins containing a GP64 “stem” domainand various portions of the GP64 ectodomain was generated. FIG. 6 showsan overall strategy for mapping GP64 regions necessary for display ofGP64 derived peptides on the BV. In one embodiment, a GP64 stem domainincluded amino acids 461-512; a total of 52 amino acids from the AcMNPVGP64 protein that included 22 residues from the predicted GP64ectodomain, the 23 residues from the predicted GP64 transmembranedomain, and the 7 residue cytoplasmic tail domain. Various portions ofthe GP64 ectodomain were fused to the GP64 stem such that in the matureprotein, the stem construct was fused to a cMyc epitope tag and either38 (residues 21-58), 66 (residues 21-86), 138 (residues 21-158), or 274(residues 21-294) amino acids from the N-terminus of the GP64ectodomain. The strategy for generation of these constructs and theirinsertion into the baculovirus genome is illustrated in FIG. 6D. Notethat dashed lines between boxes represent missing sequences.

The viruses expressing these constructs were amplified and titred inSf9^(Op1D) cells. Each virus was then used to infect Sf9 cells eitheralone, or by co-infection with the virus expressing a VSV G-stemprotein. Each construct was thus expressed either alone or in thepresence of a VSV G-stem. Infected cells (FIG. 6E) were examined forexpression of the various GP64-stem containing constructs and forexpression of a G-stem protein. FIG. 6E shows Western blot analysis ofcell extracts from cells infected with viruses expressing the G-stemconstruct or the GP64-stem fusions alone (FIG. 6E, left panel) orco-infections of viruses expressing the GP64-stem fusions and a virusexpressing the VSV G-stem construct (FIG. 6E, right panel). Small closedcircles show the positions of the fusion proteins containing theGP64-stem, and open circles show the position of a control wild typeGP64 protein containing an N-terminal cMyc epitope. An anti-cMycmonoclonal antibody was used to detect tagged proteins.

Virions from each infection were then purified and examined for thepresence of the GP64-stem constructs, and for G-stem (FIG. 6F). FIG. 6Fshows Western blot analysis of purified budded virions (BV) from cellsinfected with viruses expressing GP64-stem fusions (FIG. 6F, left panel)or co-infected with viruses expressing the GP64-stem fusions and a virusexpressing the VSV G-stem construct (FIG. 6F, right panel). Mostconstructs were readily detected in BV alone (FIG. 6F, left panel) andthe levels of GP64-stem fusions were substantially enhanced in BV thatalso contained G-stem (right panel; compare lanes 2, 3, and 5 betweenleft and right panels). An anti-cMyc monoclonal antibody was used todetect tagged proteins.

The results illustrate that most constructs were expressed well ininfected cells. In some cases, the G-stem construct was expressed atlower levels when the G-stem expressing virus was co-infected with thelonger GP64-stem constructs (FIG. 6E, lanes 4-6). However, when purifiedvirions were examined, G-stem protein was found in all virionpreparations (FIG. 6F, right panel). In all co-infections except one,GP64-stem constructs were found in the purified virion preparations andthe levels detected were in most cases substantially higher than thatdetected in the absence of a G-stem expressing virus (FIG. 6F, compareGP64 fusions in left and right panels). This indicates that a stemportion of GP64 can be used to target proteins to the virion, even whena G-stem construct is used to stimulate budding in the absence of wildtype GP64.

There are at least two separate functions of GP64 that relate to itsrole in the budding and assembly of the BV. First, GP64 is required forefficient budding. The present invention determines that a very limitedheterologous protein construct, a VSV-G stem, is capable of substitutingfor the budding function of GP64. The second functional domain ofinterest is the “targeting domain” that targets the GP64 protein forinclusion in the assembled virion.

To identify this BV targeting domain, a G-stem was used to provide thebudding function and a series of deletion constructs were generated tomap the targeting function.

Using a gp64null AcMNPV virus that expresses a G-stem from VSV, a seriesof GP64 constructs that contained either N- or C-terminal truncations ofthe ectodomain (FIGS. 6A-6C) were inserted. FIG. 6A shows a strategy forconstructing a series of cMyc tagged GP64 constructs that are truncatedat the N-terminus of the GP64 ectodomain. Each construct contained theGP64 signal peptide, signal cleavage site, and a cMyc epitope tag.Various amounts of the mature N-terminus of the GP64 ectodomain aredeleted from the constructs. Construct names are indicated on the rightof each diagram.

FIG. 6B shows Western blot analysis of cell extracts from cells infectedwith gp64null viruses expressing the GP64 constructs shown in FIG. 6A(FIG. 6B, left panel) or co-infected with the viruses expressing theconstructs from FIG. 6A and a virus expressing the VSV G-stem construct(FIG. 6B, right panel). An anti-cMyc monoclonal antibody was used todetect tagged proteins. Small closed circles show the positions of thetruncated GP64 constructs and open circles show the position of acontrol wild type GP64 protein containing an N-terminal cMyc epitope.Expression of each construct was detected in infected Sf9 cells. Anarrowhead shows the position of the G-stem protein.

FIG. 6C shows Western blot analysis of purified budded virions fromcells infected with viruses expressing the GP64 constructs shown in FIG.6A (FIG. 6C, left panel) or co-infected with the viruses expressing theconstructs from FIG. 6A and a virus expressing the VSV G-stem construct(FIG. 6C, right panel). An anti-cMyc monoclonal antibody was used todetect tagged proteins. Only the full length GP64 construct (Ntr4,21-512) was readily detected in purified virions.

The predicted GP64 ectodomain is comprised of the predicted amino acids21-482. Therefore, a series of gp64null viruses containing GP64N-terminal deletions downstream of amino acid 21 (FIG. 6A) weregenerated. Four constructs were examined. These constructs were acontrol construct that contained no GP64 ectodomain deletion and threeother constructs containing N-terminal deletions of 21-124, 21-270, and21-376 amino acids, respectively. These constructs were all expressedwell in infected Sf9 cells (FIG. 6B, Cell Extracts). When BVpreparations (from Sf9 cells infected with each construct) wereanalyzed, the full length control GP64 construct was detected inabundance, indicating robust BV production and inclusion of the GP64construct in the BV (FIG. 6C, left panel, lane 4). However, when theabove N-terminally truncated GP64 constructs were examined in a similarmanner, no GP64 or BV was detected (FIG. 6C, left panel, lanes 1-3). Inaddition, when Sf9 cells were co-infected with a) each gp64null virusexpressing an N-terminally truncated GP64 construct, and b) the gp64nullvirus expressing the G-stem construct, only the full length GP64construct was detected in the BV preparations (FIG. 6C, right panel,lane 4 vs. lanes 1-3, open circle). Substantial BV production wasdetected in each experiment that included co-infection with the gp64nullvirus expressing a G-stem construct, as determined by the detection ofthe G-stem construct in all BV preparations (FIG. 6C, right panel,arrowhead). Thus, the targeting signal appears to have been removed inall the GP64 N-terminal truncation constructs. This indicates that animportant component of the targeting signal in the native GP64 proteinis located within amino acids 21-125.

Use of the GP64-stem region to target proteins to the virion requirestwo portions of the GP64 ectodomain. When the N-terminal portion of theectodomain was deleted, and several constructs were examined for virionproduction and GP64 targeting to BV (FIGS. 6A-6C), it was found thatGP64 constructs that did not contain the N-terminal approximately 104amino acids from mature GP64, were not capable of generating detectableBV (FIG. 6C, left panel) and did not target to the BV generated fromG-stem mediated BV production (FIG. 6C, right panel). Thus, combinedwith the data from C-terminal deletion constructs (FIGS. 6D-6F), thesedata show that virion targeting by GP64 requires a portion of theN-terminus of the GP64 ectodomain (approximately <38 amino acids) plusthe C-terminal GP64-stem described above.

FIG. 6G shows a diagrammatic representation of strategies that may beused for infection or co-infection of Sf9 cells with viruses expressingfusion proteins containing the GP64-stem and display of the GP64-stemfusions on BV. The virions on the left in this figure are originallyproduced in Sf9-Op1D cells as indicated. When a gp64null virus thatexpresses either the GP64-stem or a GP64-stem fusion is used to infectSf9 cells, virion budding is partially restored. Infection of Sf9 cellswith a gp64null virus and expression of VSV G-stem results in fullrestoration of budding. In fact, the budding is robust and results inbudded virions containing VSV G stem. Co-infection with a gp64null virusexpressing VSV G-stem and a gp64null virus expressing a GP64-stem fusionoptimizes virion budding and display such that virion budding is robustand GP64-stem fusions are displayed on the budded virion envelope.

Thus, in yet another embodiment of the present invention, a heterologousgene or peptide is inserted into a construct, between a small portion ofthe N-terminus of GP64 (for example, approximately 38 amino acids of theectodomain), and a GP64-stem (the GP64 ecto-TM-CTD construct). As anexample, this construct may include: 58 amino acids from the N-terminusof GP64 (the signal peptide and 38 amino acids of the ectodomain), theheterologous protein or peptide, a small portion of the C-terminus ofthe GP64 ectodomain (such as the 22 amino acids included in the GP64stem constructs described in the studies included here) plus the GP64 TMand CTD. The N-terminal 58 amino acids of GP64 combined with a GP64-stemcould thereby rescue budding in the gp64null baculovirus.

Using small heterologous proteins or peptides in another embodiment maybe particularly useful for surface display with higher buddingefficiency, using only the above GP64-derived construction and in theabsence of wild type GP64 or a G-stem.

Targeting of proteins or peptides to budded baculovirus virions couldalso be accomplished by fusing the heterologous peptide or protein fordisplay, onto a stem region derived from a baculovirus F protein, suchas Ac23, SeF, or LdF. A suitable protein would comprise a) an N-terminalsignal peptide and the signal peptide cleavage site derived from eitherthe heterologous protein, a baculovirus protein such as GP64, or from abaculovirus F protein, b) all or a portion of the ectodomain of theheterologous peptide or protein for display, c) a moderate or smallportion of the F protein ectodomain, and d) the transmembrane domain andcytoplasmic tail domains of the F protein. Because F proteins such asOp21, Ac23, SeF, and LdF have been shown to be present in the buddedvirions of OpMNPV, AcMNPV, SeMNPV, and LdMNPV, respectively, portions ofthese F proteins or others may be used to target heterologous proteinsto the virion.

FIG. 7 shows strategies for display of heterologous proteins on thesurface of gp64null AcMNPV budded virions, as either native proteins, asfusion proteins with the VSV G-stem, or as fusion proteins with aportion of the GP64 protein (GP64-stem). This figure shows thatbaculovirus genomes with a deleted gp64 gene can be propagated asinfectious viruses, using the cell line Sf9-OP1D, which expresses theOpMNPV GP64 protein. As discussed above with respect to FIG. 2, abaculovirus genome contains a deleted gp64 gene (gp64 knockout, vAc⁶⁴⁻)and those viruses can be propagated in the cell line expressing OpMNPVGP64. When the GP64 null virus infects Sf9-Op1D, the virus without thegp64 gene can bud and propagate, thus resulting in budding of a GP64nullvirus with OpMNPV GP64 protein derived from the cell line. A geneencoding the “stem” region of the VSV G protein may be inserted into thegp64 knockout (vAc/G-Stem) and that virus propagated in the cell lineexpressing OpMNPV GP64. When the GP64null/G-stem+virus infects Sf9cells, virion budding is restored, resulting in budded virions with VSVG stem.

In another embodiment, both the gene encoding the “stem” region of theVSV G protein and a gene encoding an envelope or membrane protein areinserted into the gp64 knockout. When these virions infect Sf9 cells,VSV G stem mediates efficient budding and the resulting virions carryboth VSV-G stem and the new membrane or envelope protein. In yet anotherembodiment, both the gene encoding the “stem” region of the VSV Gprotein and a chimeric G-stem fusion are inserted into the gp64knockout. When infected into Sf9 cells, the VSV G stem mediatesefficient budding and the chimeric G-stem fusion is targeted to thevirion membrane. In still another embodiment, both the gene encoding theVSV G stem and genes encoding a chimeric GP64 fusion protein areinserted into the gp64 knockout. When used to infect Sf9 cells, VSV Gstem mediates efficient budding and the chimeric GP64 fusion is targetedto the virion membrane.

Recombinant baculoviruses expressing chimeric HA proteins wereconstructed as an example of baculovirus virion display of heterologousproteins. FIGS. 8A and 8B show enhanced targeting of HA fusions toAcMNPV BV and enhanced budding mediated by the G-stem construct.Chimeric HA-GP64 protein constructs were generated by fusing the HAectodomain (18-528) with the GP64 signal peptide and various portionsfrom the N terminus of the GP64 ectodomain, and the GP64-stem region orthe native HA TM and CTD domains. Constructs were expressed under thecontrol of the gp64 promoter. Two chimeric HA-GP64 proteins eachcontained an N-terminal cMyc epitope tag and either 38 or 66 residues ofthe GP64 ectodomain (at the N terminus), and a 91 amino acid GP64-stemsequence at the C terminus (constructs 58-HA-TM-CTD and 86-HA-TM-CTD).Construct 58-HA-FL contains a cMyc epitope tag, 38 amino acids from theN terminus of the GP64 ectodomain, and the HA ectodomain, TM, and CTD(residues 18-565).

FIG. 8B shows Western blot analysis of BV preparations. Purified BVpreparations derived from cells infected with viruses expressing HAfusion proteins (lanes 7, 8 and 9) or co-infected with virusesexpressing HA fusion proteins and a virus expressing the VSV G-stemconstruct (lanes 3, 4 and 5) were examined for the presence of envelopeprotein constructs using an anti-cMyc antibody (Purified BV, top panel)or an anti-HA antibody (Purified BV, α-HA, lower panel). VP39 frompurified ³⁵S-Methionine labeled progeny BV (Purified BV, middle panel,Labeled VP39) was detected by PhosphorImager™ analysis and used to moredirectly compare levels of progeny BV production. A control infectionwith only the virus expressing the cMyc-tagged G-stem construct is shownin lane 2. Cell extracts from the above preparations were also examinedfor protein expression using anti-cMyc antibody.

The influenza A/WSN/33 HA gene encoding the ectodomain (amino acids 18to 528) was PCR amplified from plasmid pEWSN-HA (Neumann et al., 2000,Journal of Virology 74:547-551, incorporated herein by reference). Aforward primer (Kpn-HA Forward) with a Kpn1 restriction site engineeredinto the 5′ end (SEQ ID NO: 1, see Sequence Listing; which includedsequence immediately downstream of the HA signal peptide), was used incombination with a downstream primer. The downstream primer (SEQ ID NO:2) contained a KpnI site engineered for in-frame insertion of the HAgene into vector pFB-gp64sig-cmyc-58-TM-CTD orpFB-gp64sig-cmyc-86-TM-CTD, which are pFastBac-derived plasmidscontaining the gp64 promoter, and sequence encoding the gp64 signalpeptide and cleavage site, a cMyc tag, 38 or 66 amino acids of the GP64N-terminal ectodomain and a KpnI cloning site, followed by 21 aminoacids from the GP64 C-terminal ectodomain and the GP64 TM and GP64 CTD.The PCR product was digested with Kpn1 and ligated into the Kpn1 sitesof vector pFB-gp64sig-cmyc-58-TM-CTD or pFB-gp64sig-cmyc-86-TM-CTD, togenerate constructs containing the HA ectodomain and the GP64-stem. Theresulting constructs were designated pFB-58-HA-TM-CTD andpFB-86-HA-TM-CTD, respectively. Thus, each construct expresses a proteinthat contains an N-terminal cMyc tag, a variable portion of the matureN-terminal region of the GP64 protein, the HA ectodomain and theGP64-stem region (see FIG. 8A). In addition, a construct containing theGP64 promoter and signal peptide combined with the HA ectodomain, HA TMand CTD (HA amino acids 18 to 565) was also generated by first PCRamplifying the downstream portion of the HA gene from plasmid pEWSN-HA(25) and inserting it into a pFastbac plasmid. The HA sequences wereamplified using the same forward primer (Kpn-HA Forward) in combinationwith a downstream primer (SEQ ID NO: 3). The downstream primer containedan HindIII site engineered for insertion of the HA gene into vectorpFB-gp64sig-cmyc-58-TM-CTD. The resulting plasmid pFB-58-HA-FL encodes aprotein that contains an N-terminal cMyc tag, a portion of the matureN-terminal region of the GP64 protein, the HA ectodomain and the HA TMand CTD region (FIG. 8A, 58-HA-FL).

G-stem and GP64 domains necessary for budding and virion targeting maybe effectively used together. Portions of the GP64 protein alone wereexamined to determine if they may be used to target foreign proteins togp64null virions. The ectodomain of influenza HA (A/WSN/33) was fusedbetween the C-terminal GP64-stem and various portions of the N-terminalectodomain to determine if the mapped targeting and budding domains ofGP64 were sufficient for rescue of budding and targeting of aheterologous protein to the virion (FIG. 8A). N-terminal fusionscontained the GP64 signal peptide, a cMyc epitope, and 38 or 66 aminoacids from the N terminus of the GP64 ectodomain. The C terminus of eachconstruct was comprised of either the 52 amino acid GP64 stem region(58-HA-TM-CTD; 86-HA-TM-CTD) or the wild type HA TM and CTD (FIG. 8A,58-HA-FL). Each construct was inserted into a gp64null AcMNPV genome andthe resulting viruses were propagated in Sf9^(Op1D) cells. Sf9 cellswere then infected with each virus either alone, or in combination witha virus expressing the G-stem construct. On Western blots of purified BVpreparations challenged with an anti-cMyc antibody, the HA ectodomainfusions were detected abundantly (FIG. 8B, upper panel, HA). Theidentity of HA fusions was also confirmed by Western blot analysis withan anti-HA polyclonal antiserum (FIG. 8B, Anti-HA). Co-infection of eachof the HA-fusion constructs with a virus expressing the G-stem constructresulted in higher levels of detection of the HA-fusions, and in theseconstructs the G-stem was detected at lower levels. When expressed inthe presence of the G-stem construct, two of the ectodomain fusions(58-HA-TM-CTD and 86-HA-TM-CTD) were detected at levels that appeared tobe similar to the abundant expression of the G-stem construct alone.Although the HA construct that contained its own TM and CTD domains wasexpressed and detected on the purified BV, the levels were clearly lowerthan that of either construct containing the GP64 stem region (FIG. 8B,lane 5 versus lanes 3 and 4; lane 9 versus lanes 7 and 8). Relativedifferences in budded virion production were more directly observed byexamining the relative levels of the major capsid protein (VP39) inpurified labeled budded virion preparations (FIG. 8B, Labeled VP39,compare lane 5 versus lanes 3 and 4; lane 9 versus lanes 7 and 8). Thus,the combined use of G-stem stimulated budding and the GP64 targetingdomain resulted in more efficient display of this heterologous proteinon the AcMNPV BV. Most prior studies of virion display have utilizedprotein expression from the very strong polyhedrin or p10 promoterwhereas the constructs generated in the present invention were generatedwith the native GP64 early/late promoter. Higher expression levels maylead to a higher abundance of the displayed protein on the cell surfaceafter optimizing the budding and targeting as described herein.

By generating viruses that express chimeric influenza virushemagllutinin (HA) proteins containing the GP64 targeting domain andcoinfecting those viruses with a virus expressing the G-stem construct,the present invention shows enhanced display of the HA protein ongp64null AcMNPV budded virions. The combined use of gp64null virions,VSV G-stem enhanced budding, and GP64 domains for targeting heterologousproteins to virions is useful for applications including, but notlimited to, targeted transduction of mammalian cells and vaccineproduction.

Specific examples of methods used to practice the above-describedembodiments of the invention are detailed below. Other methods, known bythose skilled in the art, could alternatively be used without deviatingfrom the spirit of the invention.

Construction of the gp64-Null AcMNPV Bacmid

The gp64 gene of an AcMNPV bacmid (bMON14272; Invitrogen) was deletedfrom the AcMNPV genome by a modification of the method taught in Bideshiand Federici, J Gen Virol 81:1593-1599, 2000, herein incorporated byreference, as reported in Lung et al. Briefly, a chloramphenicolresistance gene (cat) cassette was amplified by PCR and cloned togenerate plasmid pCh1R-CRIIblunt. The insert containing the cat cassettewas excised from pCh1R-CRIIblunt and was used to replace the SpeI-BglIIfragment (containing the gp64 gene) in pAcEcoHΔSma, a plasmid containingthe AcMNPV gp64 ORF and flanking sequences (13), resulting in generationof plasmid, pAcEcoHΔSma, gp64 (Ch1R). An EcoRI and HindIII fragment wasexcised and gel purified, then cotransformed with AcMNPV bacmidbMON14272. A colony resistant to kanamycin and chloramphenicol wasselected and analyzed and named vAc^(gp64−). The virus vAc^(gp64−) waspropagated in Sf9^(Op1D) cells which constitutively express the OpMNPVGP64 protein.

Donor Plasmids Containing G-Stem Fusion Protein Genes

To express a VSV G-Stem construct in the context of a gp64null AcMNPVvirus, a donor plasmid construct designated pFBcMyc-G-Stem wasgenerated. A truncated version of the vesicular stomatitis virus (VSV) Gprotein, containing 91 amino acides that included 42 amino acids ofC-terminal ectodomain, plus the transmembrane (TM) and cytoplasmicterminal domains (CTD) (20 amino acids and 29 amino acids,respectively), was generated by PCR-mediated mutagenesis in thefollowing manner. A forward primer with an EcoR1 restriction siteengineered into the 5′ end (SEQ ID NO: 4), was used in combination witha reverse primer that contained an XbaI site (SEQ ID NO: 5) to amplifythe “stem” portion of the VSV G gene from a wild type VSV G DNA template(pSM8141-VSV G) (11). Thus, EcoRI and XbaI restriction sites wereengineered into the 5′ and 3′ ends, respectively, of the G-Stem PCRproduct. The PCR product was digested with EcoRI and XbaI, purified, andligated into the EcoRI and XbaI sites of vector pdFB-gp64sig-cMyc, apFastBac-derived plasmid containing the gp64 promoter, the signalpeptide and cleavage site, followed by a cMyc epitope tag and a cloningsite (FIG. 1B). The resulting construct was named pFBcMyc-G-stem. Thus,the truncated form of the VSV G gene that was cloned into vectorpdFB-gp64sig-cMyc expresses a protein that contains an N-terminal cMycepitope tag linked by a Phe residue to the truncated VSV G protein.

Transpositions of inserts from donor plasmids into the gp64-null bacmidwere initially detected by gentamicin resistance and blue-whitescreening according to the BAC-to-BAC manual (Invitrogen), and furtherconfirmed by PCR analysis and by DNA sequencing. Cells stably expressingOpMNPV GP64 (cell line Sf9^(Op1D)) were transfected with each bacmid DNAand the resulting viruses were harvested from cell supernatants andtitred on Sf9^(Op1D) cells. The resulting virus was designatedvAc/G-Stem.

A series of plasmids was also constructed in which C-terminaltruncations of GP64 or the enhanced green fluorescent protein (EGFP)coding region were cloned in frame between the cMyc epitope and the VSVG Stem domains (FIGS. 1B, 5A and 5B) of plasmid pFBcMyc-G-Stem. DNAfragments encoding C-terminally truncated portions of the GP64 openreading frame were PCR amplified from a wild type (wt) AcMNPV DNAtemplate. A single forward primer with an EcoRI restriction siteengineered into the 5′ end (SEQ ID NO: 6; corresponding to a sequence 57base pairs downstream of the gp64 start codon), was used in combinationwith a downstream primer specific for each truncation (Table 1). Eachdownstream primer contained an EcoRI site engineered for in-frameinsertion of the AcMNPV gp64 gene into vector pFBcMyc-G-Stem. Thus,EcoRI restriction sites were engineered into both 5′ and 3′ ends, ofeach PCR product. Each PCR product was digested with EcoRI, purified,and ligated into the EcoRI sites of vector pFBcMyc-G-Stem, to generate atruncated GP64 ORF fused in-frame at the N-terminus of the VSV G-Stem.Each construct was confirmed by PCR to confirm the correct orientationof the insertions and also by sequencing across the junctions. Theresulting viruses were produced as described above and the viruses weredesignated: vAc/130-G-Stem, vAc/152-G-Stem, vAc/160-G-Stem,vAc/166-G-Stem, vAc/175-G-Stem and vAc/EGFP-G-Stem.

Constructs encoding N-terminal truncations of the GP64 ectodomain (seeFIG. 6A) express proteins containing an N-terminal c-Myc epitope taglinked by a Phe residue to a portion of the GP64 ectodomain truncated atthe N terminus of the mature GP64 protein.

Analysis of Progeny Virion Production by ³⁵S-Methionine Labeling

Progeny virions from infections with viruses vAc^(gp64−/Acgp64),vAc^(gp64−) or vAc/G-Stem were labeled with ³⁵S-methionine in thefollowing manner. Sf9 cells (1×10⁷ cells) were plated in a T-25 flask(Corning Inc.). After cells were allowed to attach for 1 hour, they wereinfected with recombinant virues at an MOI of 10 for 1 hour. At 29 hourspost infection, the cells were starved by incubation in 3 mlmethionine-free Grace's medium (Invitrogen) for 1 hour, followed byaddition of ³⁵S-EasyTag Express protein labeling mix (1175.0 Ci/mmol,Perkin-Elmer) to a final concentration of 10 μCi/ml. At 37 hours postinfection, unlabeled methionine was added to a final concentration of 10mM and cells were incubated at 27° C. for an additional 48 hours.Supernatants were harvested and virions purified by pelleting through a25% sucrose cushion at 100,000×g for 90 minutes at 4° C. in a BeckmanSW60 rotor. Virus pellets were resuspended in 300 μl Phosphate BufferedSaline (PBS, pH 6.2).

Construction of Plasmids and Baculoviruses Encoding ConstructsContaining a GP64-Stem Region

A series of plasmids encoding the C-terminal GP64 region (the GP64-stemregion) and varying portions of the GP64 ectodomain, was generated bythe following strategy: First, DNA fragments containing variableportions of the GP64 open reading frame were PCR amplified from a wildtype (wt) AcMNPV DNA template. A forward primer with an EcoR1restriction site engineered into the 5′ end (SEQ ID NO: 6; whichincluded sequence immediately downstream of the gp64 signal peptide),was used in combination with a downstream primer specific for eachtruncation (Table 1).

TABLE 1 PCR primers for amplification of EGFP and portions of GP64Forward Primer: SEQ ID NO: 6 Reverse Primers: 130 reverse: SEQ ID NO: 7152 reverse: SEQ ID NO: 8 160 reverse: SEQ ID NO: 9 166 reverse: SEQ IDNO: 10 175 reverse: SEQ ID NO: 11 EGFP Primers: EGFP forward: SEQ ID NO:12 EGFP reverse: SEQ ID NO: 13

TABLE 2 PCR primers for amplification of portions of GP64 ForwardPrimer: SEQ ID NO: 6 Reverse Primers: Tyr58: SEQ ID NO: 14 Arg86: SEQ IDNO: 15 Val158: SEQ ID NO: 16 Gly294: SEQ ID NO: 17

Each downstream primer contained a KpnI site engineered for in-frameinsertion of the AcMNPV gp64 gene into vector pFB-gp64sig-cmyc-TM-CTD, apFastBac-derived plasmid containing the gp64 promoter, and sequenceencoding the signal peptide and cleavage site, a cMyc tag and a cloningsite, followed by 21 amino acids of GP64 C-terminal ectodomain, plus theGP64 transmembrane (TM) and GP64 cytoplasmic tail domains (CTD). EachPCR product was digested with EcoRI and Kpn1 and ligated into the EcoRIand Kpn1 sites of vector pFB-gp64sig-cmyc-TM-CTD, to generate a seriesof constructs each containing the GP64-stem and varying portions of theGP64 ectodomain (FIG. 6). Thus, each construct expresses a protein thatcontains an N-terminal cMyc tag, a portion of the mature N-terminalregion of the GP64 protein, and the GP64-stem region. Each construct wasconfirmed by sequencing across the junctions. The resulting viruses wereproduced as described above and the viruses were designated:vAc/58-TM-CTD, vAc/86-TM-CTD, vAc/158-TM-CTD and vAc/294-TM-CTD (FIG.6B).

Co-Infections

For co-infection experiments, Sf9 cells (1×10⁵ cells) seeded on eachwell of a E-well-plate were co-infected at a total MOI of 10 with theequal infectious units of the vAc/G-Stem virus plus one of the followingviruses: vAc/58-TM-CTD, vAc/86-TM-CTD, vAc/158-TM-CTD or vAc/294-TM-CTD.Progeny virions derived from co-infection experiments were purified andanalyzed as described above. Viruses used for coinfections included thegp64null baculovirus, a control virus and those illustrated in FIGS. 6A,6D and 8A.

Western Blot Analyses

Cell lysates were prepared by washing cultured cells with phosphatebuffered saline (PBS) and resuspending cells in NET buffer (20 mM Tris,pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1.0% Nonidet P-40, 1 mM EDTA) towhich a protease inhibitor cocktail (Complete; Roche Applied Science)was added according to the manufacturer's instructions. NET buffer (500μl) was added to 1×10⁶ cells and incubated for 30 minutes at 4° C., andthen nuclei were removed by pelleting at 4° C. for 10 minutes at18,000×g. Virus purification was performed as described above. ForWestern blot analysis, 10 μl of the cell lysate or purified viruses weremixed with 10 μl of 2× Laemmli buffer (125 mM Tris, 2% sodium dodecylsulfate (SDS), 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromophenolblue, pH 6.8) and heated to 100° C. for 5 minutes prior to SDS-10%polyacrylamide gel electrophoresis (SDS-PAGE). Gels were blotted ontoImmobilon-P membranes (Millipore) and blocked overnight at 4° C. in TBST(25 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20, 5% powdered milk).Blots were incubated for 1 hour at room temperature with the followingprimary antibodies diluted in TBST: anti cMyc MAb (from hybridomasupernatant) diluted 1:1000, anti-VP39 MAb diluted 1:1000, anti-HA(chicken polyclonal antisera) diluted 1:100, or anti-VSV G (MAb P5D4)diluted 1:10,000. After washing 3 times in TBST, blots were incubatedwith a secondary antibody consisting of a goat anti-mouse IgG-alkalinephosphatase conjugate (Promega) at a dilution of 1:10,000. Western blotswere processed as described in Blissard et al., 1992, J. Virol.66:6829-6835, herein incorporated by reference.

Fluorescence Microscopy

Sf9 cells (1×10⁵ cells) seeded on 6-well-plate were infected at an MOIof 10 with the vAc/EGFP-G-Stem virus expressing EGFP-G-Stem andincubated for 24 and 48 hours. Epiflorescence microscopy was performedwith an inverted IX70 microscope (Olympus).

It will be apparent that permutations of the methods and strategiesoutlined above may be useful to rescue budding from GP64-protein-nullbaculoviruses. It will also be apparent that permutations of thedisclosed G-stem compositions and constructs thereof are possible. Usingmethods disclosed herein one may perform experiments to determineprecise quantitative differences in virion budding when constructscontaining different VSV G protein fragments are expressed in abaculovirus (or otherwise provided) and thereby identify alternateG-stem compositions and constructs suitable for the invention. The useof G-stem or GP64-stem constructs as chimeric fusions with heterologousproteins to target proteins to the virion membrane or envelope could beused separately or in combination with any of the enhanced buddingembodiments disclosed herein. Accordingly, it is to be understood thatthe embodiments of the invention herein described are merelyillustrative of the application of the principles of the invention.Reference herein to details of the illustrated embodiments is notintended to limit the scope of the claims, which themselves recite thosefeatures regarded as essential to the invention.

What is claimed is:
 1. A method of restoring efficient buddingcapability in a gp64null baculovirus where the baculovirus envelopeglycoprotein gp64 is absent in the baculovirus, comprising the step ofa) inserting a Vesicular Stomatitis Virus G-stem construct encodingamino acids 421 to 511 of a Vesicular Stomatitis Virus G-stem proteininto the viral genome of the baculovirus, thereby restoring efficientbudding capability in the baculovirus.
 2. The method of claim 1, furthercomprising, prior to step a), the step of b) knocking out the geneencoding the baculovirus envelope glycoprotein gp64 in the baculovirus.3. The method of claim 1, wherein the gp64null baculovirus is derivedfrom wild type Autographa californica multicapsid nucleopolyhedrovirusor a gp64+ Autographa californica multicapsid nucleopolyhedrovirusbacmid.
 4. The method of claim 3, further comprising, prior to step a),the step of b) knocking out a gp64 gene in the Autographa californicamulticapsid nucleopolyhedrovirus bacmid.
 5. The method of claim 1,further comprising the step of producing baculovirus virions.